Enzymatic modification of bovine growth hormone by proteolytic streptomycete cell extracts



United States Patent Delaware N0 Drawing. Filed Nov. 23, 1964, Ser. No. 413,306 1 Claim. (Cl. 195-2) This invention relates to a process of preparing modified biologically active factors of mammalian pituitary, more particularly a process of modifying somatotropic factor of bovine pituitary glands by reacting said factor with enzymatic elaboration products of strepto mycetes.

Bovine pituitary gland is known to be a source of various biologically active factors, some of which have been prepared in forms possessing specialized biological activity, for example, the growth factor, the folliclestimulating factor, the luteinizing factor, the thyrotropic factor, the crop gland-stipulating factor and the adrenocorticotropic factor. It becoming evident that these factors are complex protein and protein-like substances, investigations are constantly under way in endeavors to prepare modified biologically active products therefrom, especially such products with modified relationships of biological activity, that is, factors which possess more or less of specific activities or are free of a specific activity. Such modified factors are valuable intermediates in further processing to arrive at essentially homogeneous factors with highly specific biological activity; the eventual aim of such modifications being elucidation of structure-function relationships so that synthetic peptides and polypeptides can be prepared with highly specific biological activity. Such further processing is exemplified by the technique known as electrophoresis on polyacrylamide gel to separate more completely the components of an individual factor and by zone electro; phoresis on starch for essentially the same purpose.

It has now been found in accordance with the present invention that a somatotropic factor of bovine pituitary gland initially possessing somatotropic, lipolytic and immunological activities can be modified by a process of enzymatic hydrolysis to yield products wherein the ratios of such activities differ from those of unmodified factor. As aforesaid these modified products are valuable intermediates for further processing in the preparation and study of the activities, peptide and amino acid content of somatotropic factor of bovine pituitary glands.

, Generally described, the process comprises reacting the somatotropic factor with a proteolytic elaboration product of certain streptomycetes under controlled conditions of pH, time and temperature which usually produce some non-protein nitrogen. A pH range of about 4 to about 11, preferably about 10 is used. A temperature of about 10 C. to about C. is operable, preferably about 25 C. The reaction is then stopped after 1 to 6 hours by the addition of a protein precipitating agent, for example, aqueous trichloracetic acid 5% to 20% concentration, or sodium tungstate in 5% trichloracetic acid. The precipitate is separated, e.g., by centrifugation and recovered. The recovered material can be purified by dissolving in alkali and dialyzing against purified water. The dialyzed solution is adjusted as to pH and applied to a column of cross-linked dextran gel which has been previously equilibrated with sodium carbonatebicarbonate aqueous buffer. Absorbed material is removed from the gel by fractional elution with the buffer, the protein content of the fractional eluates being determined by readings of optical densities by methods known in the art, for example at 280 m l in a Beckman D.U. spectrophotometer against a water blank. As peaks of protein content appear in the fractional eluates, the eluates corresponding thereto are combined, freed of buffer by dialysis against purified water, and concentrated to dryness by lyophilization. The protein corresponding to the various peaks is recovered as a dry powdery maten'al.

Assays for somatotropic and lipolytic activity and determination' of immunological response on the starting somatotropic factor of bovine pituitary and on the recovered dry material show that significant modifications of such activities have occurred.

As used herein bovine somatotropic factor means the factor prepared from bovine glands according to Wilhelmi, Hypophyseal Growth Hormone, Nature and Actions (R. W. Smith et al., eds.), pp. 59-69 (1955), and further purified as described by Reusser, Arch. Biochem. Biophys. 106:410, (1964).

The streptomycetes used in the instant process have been designated Streptomyces paliidus Sp. N., Streptomyces moderatus Sp. N., Streptomyces regalis, Sp. N., and Streptomyces rosa, Sp. N. A culture of each living organism can be obtained from the permanent collection of the Northern Utilization Research and Development Division, Agriculture Research Service, Peoria, 111., U.S.A. The respective accession numbers in this repository are NRRL No. 3149, NRRL No. 3150, NRRL No. 3151, and NRRL No. 3152.

Appearance and microscopic characterizations of the organisms are given in Tables I and II.

TABLE I.APPEARANCE OF CULTURES ON EKTACHROME 1 Agar Medium Bennett's Czapeks Sucrose Maltose-Tryptone Peptone-Iron 0.1% Tyrosine Casein Starch Culture I Surface Reverse Surface Reverse Surface Reverse Surface Reverse Surface Reverse Surface Reverse S. pallidus Gray- Brown; Gray- Yellow- Fair Brown None -Brown Trace Tan Pink Tan.

pink. pink. tan. grayypink. pink. S.m0deratus- Trace Yellow Pink Ycllow Gray- Tan "do"-.. Yell0w -do Pale Gray- Pinkpink. pink. pinkpink. tan.

. an. S.regalis Bluc Brown; Trace Brown Blue Brown No aerial. Brown Blue Tan- Trace Tanblue. brown blue. brown. 8.10m Salmond0.. Salmon- Yell0w. Salmon- "-60"--. Trace Yellow Trace ed Salmon- Yellowsalmon. salmon tau.

1 Dietz, A., Ektaehrome Transparencies as Aids in Actinomycete Classification, Annals of the NY. Academy of Science, 60:152-154, 1954.

TABLE IL-MIC ROSCOPIC CHARACTERISTICS S pallidus S. moderatus S. regalis S. rosa Light microscope S, RA RF Short R A, S to S, RA.

(Sporophore type). questionalble BIV-S.

Electron microscope (Spores Direct Smooth Smooth. Warty Smooth. Carbon Replica... Smooth rectangles, basket- Smooth rectangles, basket- Warty Smooth, short rectangles (boxweave surface pattern, some weave surface pattern, like), basket-weave pattern ridging. ridging. nucleus.

Utilization of carbon compounds in a synthetic medium is given in Table III.

TABLE III.-UTILIZATION OF CARBON COMPOUNDS IN A SYNTHETIC MEDIUM" S. pallz'dus S. moderatus S. regalis S. rosa Control. D-xylose. L-arabinose. Rharnnosm D-fructose D-galactose D-glucose- D-mannose Maltese Sucrose Lactose Cellobiose- Rafilnose Dextrin Inulin Soluble starch Glycerol Duleito1. D-mannitol. D-sorbitol.-. Inositoi Salicin. Phenol Cresol Na formate- Na oxalate- Na tartrate. Na salicylate. Na acetate- Na citrate" Na suceinate +=Utilization.

(+) =Utilization, slight growth. =Slight growth, no utilization.

Medium, J. Bact., 56:107-114, 1948.

Carbon compounds in Synthetic Cultural and physiological characteristics are given in Table IV.

TABLE IV.-CULTURAL AND PHYSIOLOGICAL CHARACTERISTICS Culture Agar Medium S. pallidus S. moderatus S. regalis S. rosa Peptone-iron:

Surface Gray-pink Gray-pink-tan Nn'n Pink.

Reverse Brown. Orange-tan Brown Rose-tan.

Other Melanin Melanin Melanin Melanin. Calcium malate:

Surface Cottony pink Gray-pink-tan Trace white Tan.

Reverse Cream Pale pink-tan Gray-blue Pink-tan.

Non N N n None. Malate not solubilized Malate slightly solubilized. Malate solubilized Malate not solubilized.

Glucose asparagine.

Surface Pink-white- None to slight trace pink None Pink to tan.

Rever Cream- T n T Orange-tan.

Pigment Pale yellow- Tan- T Do. Skim milk:

Surface Trace gray Very slight trace pink Yellow-tan Trace pink-tan.

Rever Tan Tan fi Orange-tan.

Pigmcnt do t .do Do. T Other Casein not solubilized Casein solubilized Casein not solubilized Casein solubilized.

yrosrne:

Surface Trace pink Pink- Non Pink-tan.

Reverse Brown do Brown Orange-tan.

Other t Tyrosine slightly solubilized. Tyrosine solubilized. Tyrosine solubilized Tyrosine not solubilized.

TABLE IV.Contiuued Culture Agar Medium S. pallidus S moderatus S. regalis S. rosa Xanthine:

Surface Trace pale pin.k Pink-tan None Pink.

Reverse Pale yellow-tando Tan Pale tan.

Pigment do Pale pi11k do Do.

Xanthme solubilized Xanthine solubilized Xanthine solubilized Xanthine solubilized. Cottony pink Pink Pale blue-tan Pink-tan. Pale yellow-tan do do Do.

Pigment-.. o None None None.

Yeast extract, malt extract:

Surface Cottony pink Fair pink-tan Very slight trace white Rose-tan.

Reverse Tan-brown Tan Tan Brown.

P1gment Tan do do Brown-tan.

Bennetts: 1

Surface ba (g+r n), pinkish white.. 5 ba (g-l-m), pinkish white 13 ba (g), bluish white 2 cc (g), grayish yellow.

Reverse 5 1e (g), light olive brown; to 4 ie (g), light brown; to 4 lg ml (g), dark grayish blue Do 13 p1 (g), deep yellowish (g), moderate brown. to dark grayish purple.

rown. Pigment--- None None 10 ml (g), dark grayish None.

blue to dark grayish purple. Opt1mumtemperature 2437 C 2428 C 2837 C 2437 C. Czapeks sucrose:

Surface a (g+m), white 3 ha (in) 2 10 ml (g), dark grayish 4 ec (g), brownish pink.

blue and dark grayish p p to 5 p brownish gray.

Reverse 2 go (g+m), grayish 31e (g), strong yellowish 9 pm (In), dark purplish Do.

yellow. brown. gray; to 8 pn (g), dark grayish reddish brown. Pigment None 8 pm (g), dark grayish None.

, reddish brown. Optimum temperature 24-37 C-.- 2428 C 2837 C 2437 C. Maltose-Tryptone: 1

Surface a (g+m), white 5 ba (g-l-m), pinkish white ch (g), light bluish gray; 2 ch (g), yellowish gray.

to 1214 ca (g), very pale purp e.

Reverse 3 pi (g), deep yellowish 5 ni (g), moderate reddish 5 pn (m), brownish gray; to 4 ni (g), moderate brown;

brown; to 3 p11 (in), dark brown. 5 nl (g), dark grayish to 3 lg (g), moderate grayish yellowish brown. brown. yellowish brown.

Pigment None None 31% (g), moderate olive 2 lg (In), light olive brown.

rown.

Optimum temperature.-- 37 C C. 37 C Z P-37 C.

Gelatin:

Brown, top 4 or medium." Pink-tan, top 34 of medium. Brown Trace tan. Complete Complete Complete Complete. Brown, top 4 of medium None Brown Trace tan. Complete Complete Complete Complete. Broth White aerial growth Trace white aerial growth. Trace white aerial growth.-- No aerial growth. Flocculent bottom growth Loose bottom growth Tan throughout and at base Colprless vegetative at ase. Tan None Tan None.

No reduction Reduction Reduction N 0 reduction. Surface Good cottony white aerial Trace white aerial growth Pale lavender-white aerial Pale pink aerial on surgrowth. growth. face pelliele. Vegetative Compact t0 floceulent Loose bottom growth Pale lavender at base Trace throughout; slight bottom growth. compact bottom growth.

Pigment. None None Pale lavender None. Other No reduction No reduction Reduction N 0 reduction. Litmus milk:

Surface Trace white aerial gr0wth Trace white aerial growth Trace ctglttony white aerial No aerial growth.

grow Peptonization None Positive Positive. Trace. p 6.1."- 7.7 7 6.1.

1 Color on Bennetts, Czapeks Sucrose, and Maltese-T 1948, and redefining in terms of the color descriptions in the No ISCC-NBS color.

ryptone was determined b ISCC-NBS Meth y using color chips from the Color Harmony Manual, 3rd Ed., 0d of Designating Colors and Dictionary of Color Names, NBS Circular Cultures of the respective streptomycetcs were grown in shake flasks in a sterile medium of pH 7.2, containing gm., cerelose and 25 gm. cottonseed flour, industrial grade (Pharmamedia, Traders Oil Mill Company, Fort Worth, Texas, U.S.A.) per liter of tap water. The flasks were inoculated with a small loop of spore material from a soil stock and incubated for 5 days at 28 C. At this time the mycelia were harvested as filter cakes and the filter cakes were washed thoroughly with purified water U.S.P. The washed cakes were :frozen at -20 C. and re-thawed to facilitate cell breakage.

Three hundred gm. of wet cake was resuspended in purified water, 600 ml. of dry glass beads was added and the total volume was made up to 1200 ml. with more purified water. The whole was homogenized in a colloid mill and the homogenate was centrifuged at about 2 C. to yield a clear supernatant. This supernatant was frozen, dried from the frozen state to a powder, which is stored at 2 C. As required for use in the inventive process, 5 mg. of the dry powder was dissolved in 5 ml. of 0.05 M sodium carbonate-bicarbonate buffer of pH 10.

How to perform the inventive process and the best mode contemplated by the inventor of carrying out the invention are set forth below for exemplification only, and not for limitation of the scope of the invention.

Somatotropic activity was determined by the rat tibia assay. Greenspan et al., Endocrinology, 452455 (1949). Lipolytic activity was determined in the epidydimal rat pad system. Marshal and Engel, J. Lipid Res. 1:339 (1960). Bovine growth hormone antiserum was prepared and tested for specificity as described by Moudgal and Li, Arch. Biochem. Biophys, 93:112 (1961).

EXAMPLE 1 Two hundred mg. bovine somatotropic factor was dissolved in 15 ml, 0.05 M sodium carbonate-bicarbonate buffer of pH 10. Enzymatic hydrolysis was started by the addition of 5 ml. of enzyme solution (1 mg. of dry enzyme prepared from Streptomyces moderatus Sp. N., above identified, dissolved in each ml. of 0.05 M-carbonate buffer of pH 10). Hydrolysis proceeded at room temperature with slow stir-ring for about one. hour, after which time the hydrolysis was stopped by the addition of an equal volume of 10% aqueous trichloracetic acid (2 C.). The whole was allowed to stand at about 2 280 m in a Beckman D.U. spectrophotometer against a water blank. Fractional eluates corresponding to various peaks of optical density were combined, dialyzed overnight .in cellophane tubing against running deionized water, and the dialyzed solution was concentrated to dryness by lyophilization to white, fluffy powders. In the exepriment three sequential peaks yielded powders 1, 2 and 3, respectively.

The biological and immunological activities of the C. for one hour and was then centrifuged at room tem- 10 powders were as follows:

\ Somatotropic Assay Lipolytic Assay Qualitative Powder Immuu. N0. of Dose/ Tibia N0. of Dose} uM FFA Assay 1 Rats rat, Width Rats rat, per 100 mg.

pg. N pg. N and 3 hr.

5 40 195 2 10 Peak 1 i 5 10 19g 3 10 i 4 26 10 Peak 2 i 4 10 135 2 10 ,i Peak 3 3 40 186 2 1O 46 Bovine Growth Hormone g g 1,92 Control 183 6 None 21 l ++=str0ng band; +=weak band.

EXAMPLE 2 perature. The supernatant contained nitrogen equivalent to 6% of the initial total nitrogen. The precipitate was recovered and dissolved in 19 ml. 0.5 N sodium hydroxide. The solution was dialyzed in cellophane (Visking dialysis cellophane tube) for approximately six hours against running cold deionized water. This dialyzed solution was adjusted to about pH with solid NaHCO and the resultant solution was applied to a column of cross-linked dextran gel having a water regain value of 10 gm./per/ gm. of dry weight (Sephadex G-lOO, Pharmacia, Uppsala, Sweden).

The column was prepared with gel of a bead size great- Following the procedure of Example 1, another 200 mg. of bovine somatotropic factor was enzymatically hydrolyzed with 5 ml. of enzyme solution prepared as hereto- 30 fore described from Str ptomyc s pallidus, Sp. N. Em

zymatic hydrolysis was allowed to proceed for 6 hours. The supernatant contained nonprotein nitrogen equivalent to 3% of the initial total nitrogen.

Again, dry powders were obtained by lyophilization of the combined fractional eluates, collected as in Example 1. The biological and immunological activities of the powders were as follows:

\ Somatotropic Assay Lipolytie Assay Qualitative Powder Irnmun. No. of Bose] Tibia No. of Bose] M FFA Assay 1 Rats rat, Width Rats rat, per 100 mg.

pg. N ug. N and 3 hr.

3 184 2 10 Peak 1 0 2; 10 i 0 10 i 5 10 209 2 10 l 56 Std 3 i8 321 g 13 1. 92 Control 5 183 6 None 21 1 ++=strong band; +=weak baud.

EXAMPLE 3 er than 180 mesh, which had been washed with 0.05 M sodium carbonate-bicarbonate buffer of pH 10. The dimensions of the column were diameter, 2.8 centimeter X height, centimeter. The column was equilibrated with the same buffer before addition of the above-mentioned dialyzed solution.

Elution was carried out with the sodium carbonatebicarbonate buffer. Fractional eluates of 10 ml. were collected and the optical densities thereof were read at Two hundred additional mg. of bovine somatotropic factor was processed according to the above directions,

except that the enzyme was that obtained from Streptomyces rosa, Sp. N., and the period of hydrolysis was four hours, after which time it was stopped by the addition of the cold trichloracetic acid solution. The supernatant did not contain nonprotein nitrogen.

The biological and immunological activities of the powders were as follows:

Somatotropie Assay Lipolytic Assay Qualitative Powder Immun. No. of Dose] Tibia No. 0! Dose/ M FFA Assay 1 Rats rat, Width Rats rat. per mg.

11g. N pg. N and 3 hr.

5 40 188 2 10 l 2 i8 is a 10} 10 Peak 2 1g 26% 2 10 1. 05 std 3 32,, 3 13 1.92 Control 5 183 6 None 21 1 ++=strong band; +=weak band.

9 EXAMPLE 4 An additional 200 mg. of bovine somatotropic factor was processed according to the aforesaid directions, ex-

Acid hydrolyses of samples were carried out in constant boiling hydrochloric acid for 22 and 72 hours at 110 C.

cept that the enzyme used was obtained from Strep- 5 in an evacuated sealed tube according to the method of tomyces regalis, Sp. N., and the period of hydrolysis was Moore et al., Methods Enzymol, 6, 819 (1963). Amino two hours. The supernatant contained nonprotein nitroacid compositions of the hydrolysates were determined gen equivalent to 4% of the initial total nitrogen. in an automatic Spinco amino acid analyzer as described The biological and immunological activities of the soby Spackman et -al., Anal. Chem., 30, 1190 (1958). The prepared powders were as follows: results were as follows:

Somatotropic Assay Lipolytic Assay Qualitative Powder Immun. No. of Dose/ Tibia No. of Dose/ 1.1M FFA Assay 1 Rats rat, Width Rats rat, per 100 mg.

14g. N g. N and 3 hr.

5 40 151 2 10 Peak 1 i g 1 age 3 10 i 0 Peak? 3 418 560 g 10 7 5 07 10 3 10 274 2 10 i Control 5 183 6 None 21 1 ++=strong band; +=weak band.

f p biqlqgiwl and T TABLE.- RELATIVE AMINO ACID COMPOSITION OFBGH- immunological activity tests FRAGMENTS EXPRESSED IN MOLAR RATIOS I 14 509 14,403 10 14,70 The biological data in the above examples show that Amino Acid 9 6 BGH the powders from the first peaks of eluates did not retain 40 1 2 3 1 2 1 2 1 2 somatotropic activity. Except for the powder of the first peak in Example 4, none retained significant lipolytic 18 2s 18 24 23 24 25 activity. Each powder reacted immunologically positive 13 5 7 6 9 8 14 8 withbovine growth hormone anti-Serums, but generally 25 21 26 22 21 23 21 25 45 32 37 33 39 38 43 33 to a weaker extent than unmodified material. All peak 2 31 23 27 23 36 28 31 26 powders retained significant but varying degrees of so- 2? i 2 g2 g8 matotropic and lipolytic activities; and immunological ac- 17 14 15 12 21 15 20 1 32222622272629 22 tivities were indistinguishable from ureacted bovine soma 45 29 35 30 33 41 31 totropic factor. 13 i4 16 i4 19 15 19 16 12 7 7 5 l3 5 s s 11 14 i4 i4 14 14 13 14 52 52 52 52 52 52 52 52 18 10 14 9 17 13 14 12 Polyacrylamlde gel electrophoresis 32 24 29 25 31 2s 2s 25 in Examples 2, 3, and 4 showed four similar patterns when compared with each other. All indicated the presence of 4 bands increasing in density from the cathode to the anode. There also existed a similarity to the pattern obtained with unhydrolyzed somatotropic factor. However, the latter yielded an electropherogram with five bands decreasing gradually in intensity from the cathode to the anode. The electropherogram from peak 2 of Example 1 differed significantly from that of the unmodified somatotropic factor.

The above amino acid compositions are expressed in molar ratios related to 52 leucine residues as observed in unprocessed bovine somatotropic factor. The data show that despite the minor differences among all fractions, none showed an amino acid spectrum significantly different from unreaoted bovine somato-tropic factor. All the fractions had a high content of the acidic amino acids, leucine and valine and had almost constant values for isoleucine.

The powders from all first peaks were found to have apparent molecular weights exceeding that of the starting growth hormone by a factor of at least 2. All powders had significant sequestering capacities for ions especially for sodium and magnesium, ash content being above 50 percent in some instances.

What is claimed is:

A process of preparing modified biologically active factors of mammalian pituitaries which comprises:

(1) reacting bovine somatotropic factor with a proteolytice elaboration product of a member selected from the group consisting of Streptomyces moderazus,

11 Sp. N.; Streptomyces pallidus, Sp. N.; Streptomyces rosa, Sp. N.; and Streptomyces regalis, Sp. N. at a pH of from about 4 to about 11 for from about 1 to .about 6 hours at a temperature of from about 10 C. to about 30 C.;

(2) adding a protein precipitating agent to the reaction mixture to form a precipitate;

(3) separating and recovering the precipitate;

(4) dissolving the precipitate in aqueous sodium hydroxide and dialyzing the solution against purified water;

(5) adjusting the Ph of the dialyzed solution to about pH and applying the adjusted solution to a column of cross-linked dextran gel having a water regain value of about 100 gm. per gm. equilibrated with 0.05 M sodium carbonate-bicarbonate aqueous buffer of pH about 10;

(6) fractionally eluting absorbed material from the gel with 0.05 M sodium carbonate-bicarbonate aqueous buffer of pH about 10;

(7) combining fractions of eluate corresponding to peaks of protein in the eluate; and

(8) recovering as dry powders modified biologically active factors from said combined fractional eluates by volatilizing water therefrom in vacuo, said powders demonstrating modified ratios of lipolytic, somatotropic and immunological activity.

References Cited by the Examiner UNITED STATES PATENTS 3,127,327 3/1964 Nomo-to et al. -62

1956, vol. 218, pp. 4152.

A. LOUIS MONACELL, Primary Examiner. L. M. SHAPIRO, Assistant Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE: OF CORRECTION Patent No. 3,317,400 May 2, 1967 Fritz Reusser It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 1, line 22, for "gland-stipulating" read g1and stimulating column 9, line 48,. for "ureacted" read unreacted column 10, line 74, for "proteolytice" read proteolytic column 11, line 11, for "Ph" read pH line 15, for "100 Gm." read 10 Gm.

Signed and sealed this 21st day of November 1967.

(SEAL) Attest:

EDWARD J. BRENNEI? Edward M. Fletcher, Jr.

Commissioner of Patents 

